cIAP1-CARD-RING mediates proteasomal-dependent down-regulation of RING-bearing IAPs. (A) HeLa cells were transfected with 6myc fusion proteins of different IAPs and with cIAP1-CR or cIAP1-CR-H588A. After 24 h of transfection, proteins were extracted, separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting with anti-myc for the detection of 6myc-IAP, anti-FLAG for the detection of cIAP1-CR, and anti-GAPDH. (B–E) HeLa cells were transfected with plasmids encoding 6myc-cIAP1 (B), cIAP2 (C), XIAP (D), or Livin (E) along with cIAP1-CR or cIAP1-CR-H588A. After 19 h of transfection, cells were treated with 2 μM proteasomal inhibitor MG132 or vehicle for an additional 5 h. The inclusion of MG132 protected IAPs from cIAP1-CR–mediated degradation.